Gene cluster involved in safracin biosynthesis and its uses for genetic engineering

ABSTRACT

A gene cluster has open reading frames which encode polypeptides sufficient to direct the synthesis of a safracin molecule.

FIELD OF THE INVENTION

The present invention relates to the gene cluster responsible for thebiosynthesis of safracin, its uses for genetic engineering and newsafracins obtained by manipulation of the biosynthesis mechanism.

BACKGROUND OF THE INVENTION

Safracins, a family of new compounds with a potent broad-spectrumantibacterial activity, were discovered in a culture broth ofPseudomonas sp. Safracin occurs in two Pseudomonas sp. strains,Pseudomonas fluorescens A2-2 isolated from a soil sample collected inTagawagun, Fukuoka, Japan (Ikeda et al. J. Antibiotics 1983,36,1279-1283; WO 82 00146 and JP 58113192) and Pseudomonas fluorescensSC 12695 isolated from water samples taken from the Raritan-DelawareCanal, near New Jersey (Meyers et al. J. Antibiot. 1983, 36(2),190-193). Safracins A and B, produced by Pseudomonas fluorescens A2-2,have been examined against different tumor cell lines and has been foundto possess antitumor activity in addition to antibacterial activity.

Due to the structural similarities between safracin B and ET-743safracin offers the possibility of hemi-synthesis of the highlypromising potent new antitumor agent ET-743, isolated from the marinetunicate Ecteinascidia turbinata and which is currently in Phase IIclinical trials in Europe and the United States. A hemisynthesis ofET-743 has been achieved starting from safracin B (Cuevas et al. OrganicLett. 2000, 10, 2545-2548; WO 00 69862 and WO 01 87895).

As an alternative of making safracins or its structural analogs bychemical synthesis, manipulating genes of governing secondary metabolismoffer a promising alternative and allows for preparation of thesecompounds biosynthetically. Additionally, safracin structure offersexciting possibilities for combinatorial biosynthesis.

In view of the complex structure of the safracins and the limitations intheir obtention from Pseudomonas fluorescens A2-2, it would be highlydesirable to understand the genetic basis of their synthesis in order tocreate the means to influence them in a targeted manner. This couldincrease the amounts of safracins being produced, because naturalproduction strains generally yield only low concentrations of thesecondary metabolites that are of interest. It could also allow theproduction of safracins in hosts that otherwise do not produce thesecompounds. Additionally, the genetic manipulation could be used forcombinatorial creation of novel safracin analogs that could exhibitimproved properties and that could be used in the hemi-synthesis of newecteinascidins compounds.

However, the success of a biosynthetic approach depends critically onthe availability of novel genetic systems and on genes encoding novelenzyme activities. Elucidation of the safracin gene cluster contributesto the general field of combinatorial biosynthesis by expanding therepertoire of genes uniquely associated with safracin biosynthesis,leading to the possibility of making novel precursors and safracins viacombinatorial biosynthesis.

SUMMARY OF THE INVENTION

We have now been able to identify and clone the genes of safracinbiosynthesis, providing the genetic basis for improving and manipulatingin a targeted manner the productivity of Pseudomonas sp., and usinggenetic methods, for synthesising safracin analogues. Additionally,these genes encode enzymes that are involved in biosynthetic processesto produce structures, such as safracin precursors, that can form thebasis of combinatorial chemistry to produce a wide variety of compounds.These compounds can be screened for a variety of bioactivities includinganticancer activity.

Therefore in a first aspect the present invention provides a nucleicacid, suitably an isolated nucleic acid, which includes a DNA sequence(including mutations or variants thereof, that encodes non-ribosomalpeptide synthetases which are responsible for the biosynthesis ofsafracins. This invention provides a gene cluster, suitably an isolatedgene cluster, with open reading frames encoding polypeptides to directthe assembly of a safracin molecule.

One aspect of the present invention is a composition including at leastone nucleic acid sequence, suitably an isolated nucleic acid molecule,that encodes at least one polypeptide that catalyses at least one stepof the biosynthesis of safracins. Two or more such nucleic acidsequences can be present in the composition. DNA or corresponding RNA isalso provided.

In particular the present invention is directed to a nucleic acidsequence, suitably an isolated nucleic acid sequence, from a safracingene cluster comprising said nucleic acid sequence, a portion orportions of said nucleic acid sequence wherein said portion or portionsencode a polypeptide or polypeptides or a biologically active fragmentof a polypeptide or polypeptides, a single-stranded nucleic acidsequence derived from said nucleic acid sequence, or a single strandednucleic acid sequence derived from a portion or portions of said nucleicacid sequence, or a double-stranded nucleic acid sequence derived fromthe single-stranded nucleic acid sequence (such as cDNA from mRNA). Thenucleic acid sequence can be DNA or RNA.

More particularly, the present invention is directed to a nucleic acidsequence, suitably an isolated nucleic acid sequence, which includes orcomprises at least SEQ ID 1, variants or portions thereof, or at leastone of the sacA, sacB, sacC, sacC, sacD, sacE, sacF, sacG, sacH, sacH,sacI, sacJ, orf1, orf2, orf3 or orf4 genes, including variants orportions. Portions can be at least 10, 15, 20, 25, 50, 100, 1000, 2500,5000, 10000, 20000, 25000 or more nucleotides in length. Typically theportions are in the range 100 to 5000, or 100 to 2500 nucleotides inlength, and are biologically functional.

Mutants or variants include polynucleotide molecules in which at leastone nucleotide residue is altered, substituted, deleted or inserted.Multiple changes are possible, with a different nucleotide at 1, 2, 3,4, 5, 10, 15, 25, 50, 100, 200, 500 or more positions. Degeneratevariants are envisaged which encode the same polypeptide, as well asnon-degenerate variants which encode a different polypeptide. Theportion, mutant or variant nucleic acid sequence suitably encodes apolypeptide which retains a biological activity of the respectivepolypeptide encoded by any of the open reading frames of the safracingene cluster. Allelic forms and polymorphisms are embraced.

The invention is also directed to an isolated nucleic acid sequencecapable of hybridizing under stringent conditions with a nucleic acidsequence of this invention. Particularly preferred is hybridisation witha translatable length of a nucleic acid sequence of this invention.

The invention is also directed to a nucleic acid encoding a polypeptidewhich is at least 30%, preferably 50%, preferably 60%, more preferably70%, in particular 80%, 90%, 95% or more identical in amino acidsequence to a polypeptide encoded by any of the safracin gene clusteropen reading frames sacA to sacJ and orf1 to orf4 (SEQ ID 1 and genesencoded in SEQ ID 1) or encoded by a variant or portion thereof. Thepolypeptide suitably retains a biological activity of the respectivepolypeptide encoded by any of the safracin gene cluster open readingframes.

In particular, the invention is directed to an isolated nucleic acidsequence encoding for any of SacA, SacB, SacC, SacD, SacE, SacF, SacG,SacH, SacI, SacJ, Orf1, Orf2, Orf3 or Orf4 proteins (SEQ ID 2-15), andvariants, mutants or portions thereof.

In one aspect, an isolated nucleic acid sequence of this inventionencodes a peptide synthetase, a L-Tyr derivative hidroxylase, a L-Tyrderivative methylase, a L-Tyr O-methylase, a methyl-transferase or amonooxygenase or a safracin resistance protein.

The invention also provides a hybridization probe which is a nucleicacid sequence as defined above or a portion thereof. Probes suitablycomprise a sequence of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, ormore nucleotide residues. Sequences with a length on the range 25 to 60are preferred. The invention is also directed to the use of a probe asdefined for the detection of a safracin or ecteinascidin gene. Inparticular, the probe is used for the detection of genes inEcteinascidia turbinata.

In a related aspect the invention is directed to a polypeptide encodedby a nucleic acid sequence as defined above. Full sequence, variant,mutant or fragment polypeptides are envisaged.

In a further aspect the invention is directed to a vector, preferably anexpression vector, preferably a cosmid, comprising a nucleic acidsequence encoding a protein or biologically active fragment of aprotein, wherein said nucleic acid is as defined above.

In another aspect the invention is directed to a host cell transformedwith one or more of the nucleic acid sequences as defined above, or avector, an expression vector or cosmid as defined above. A preferredhost cell is transformed with an exogenous nucleic acid comprising agene cluster encoding polypeptides sufficient to direct the assembly ofa safracin or safracin analog. Preferably the host cell is amicroorganism, more preferably a bacteria.

The invention is also directed to a recombinant bacterial host cell inwhich at least a portion of a nucleic acid sequence as defined above isdisrupted to result in a recombinant host cell that produces alteredlevels of safracin compound or safracin analogue, relative to acorresponding nonrecombinant bacterial host cell.

The invention is also directed to a method of producing a safracincompound or safracin analogue comprising fermenting, under conditionsand in a medium suitable for producing such a compound or analogue, anorganism such as Pseudomonas sp, in which the copy number of thesafracin genes/cluster encoding polypeptides sufficient to direct theassembly of a safracin or safracin analog has been increased.

The invention is also directed to a method of producing a safracincompound or analogue comprising fermenting, under conditions and in amedium suitable for producing such compound or analogue, an organismsuch as Pseudomonas sp in which expression of the genes encodingpolypeptides sufficient to direct the assembly of a safracin or safracinanalogue has been modulated by manipulation or replacement of one ormore genes or sequence responsible for regulating such expression.Preferably expression of the genes is enhanced.

The invention is also directed to the use of a composition including atleast one isolated nucleic acid sequence as defined above or amodification thereof for the combinatorial biosynthesis of non-ribosomalpeptides, diketopiperazine rings and safracins.

In particular the method involves contacting a compound that is asubstrate for a polypeptide encoded by one or more of the safracinbiosynthesis gene cluster open reading frames as defined above with thepolypeptide encoded by one or more safracin biosynthesis gene clusteropen reading frames, whereby the polypeptide chemically modifies thecompound.

In still another embodiment, this invention provides a method ofproducing a safracin or safracin analog. The method involves providing amicroorganism transformed with an exogenous nucleic acid comprising asafracin gene cluster encoding polypeptides sufficient to direct theassembly of said safracin or safracin analog; culturing the bacteriaunder conditions permitting the biosynthesis of safracin or safracinanalog; and isolating said safracin or safracin analog from said cell.

The invention is also directed to any of the precursor compounds P2,P14, analogs and derivatives thereof and their use in the combinatorialbiosynthesis non-ribosomal peptides, diketopiperazine rings andsafracins.

Additionally, the invention is also directed to the new safracinsobtained by knock out safracin P19B, safracin P22A, safracin P22B,safracin D and safracin E, and their use as antimicrobial or antitumoragents, as well as their use in the synthesis of ecteinascidincompounds.

The invention is also directed to new safracins obtained by directedbiosynthesis as defined above, and their use as antimicrobial orantitumor agents, as well as their use in the synthesis of ecteinascidincompounds. In particular the invention is directed to safracin B-ethoxyand safracin A-ethoxy and their use.

In one aspect, the present invention enables the preparation ofstructures related to safracins and ecteinascidins which cannot or aredifficult to prepare by chemical synthesis. Another aspect is to use theknowledge to gain access to the biosynthesis of ecteinascidins inEcteinascidia turbinata, for example using these sequences or parts asprobes in this organism or a putative symbiont.

More fundamentally, the invention opens a broad field and gives accessto ecteinascidins by genetic engineering.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Structural organization of the chromosomal DNA region cloned inpL30p cosmid. The region of P. fluorescens A2-2 DNA, containing thesafracin gene cluster, is shown. Both, sacABCDEFGH and sacIJ, geneoperons and the modular organization of the peptide synthetases deducedfrom sacA, sacB and sacC are illustrated. The following domains areindicated: C: condensation; T: thiolation; A: adenylation and Re:reductase. Location of other genes present in pL30p cosmid (orf1 toorf4) as well as their proposed function is shown.

FIG. 2: Conserved core motifs between NRPSs. Conserved amino acidsequences in SacA, SacB and SacC proteins and their comparison with itshomologous sequences from Myxococcus xanthus DM50415.

FIG. 3. NRPS biosynthesis mechanism proposed for the formation of theAla-Gly dipeptide. Step a*, adenylation of Ala; b*, transfer to the4′-phosphopantetheinyl arm; c*, transfer to the waiting/elongation site;d*, adenylation of the Gly; e*, transfer to the 4′-phosphopantetheinylarm; f*, condensation of the elongation chain on the4′-phosphopantetheinyl arm with the starter chain at thewaiting/elongation site; g*, Ala-Gly dipeptide attached to thephosphopantetheinyl arm of SacA and h*, transfer of the elongated chainto the following waiting/elongation site.

FIG. 4: Cross-feeding experiments. A. Scheme of A2-2 DNA fragmentscloned in pBBR1-MCS2 vector and products obtained in the heterologoushost. B. HPLC profile of safracin production in wild type strain versussacF mutant. The addition of P2 precursor to the sacF mutant, providedboth in trans and synthetically, yield safracin B production. SfcA,safracin A and SfcB, safracin B.

FIG. 5: Scheme of the safracin biosynthesis mechanism and biosyntheticintermediates. Single enzymatic steps are indicated by a continuousarrow and multiple reactions steps are indicated by discontinuousarrows.

FIG. 6: Safracin gene disruptions and compounds produced. A. Genedisruption and precursor molecules synthesized by the mutantsconstructed. Gene marked With an asterisk does not belong to thesafracin cluster. Inactivation of genes orf1, orj2, orf3 and orf4 hasdemonstrated to have no effect over safracin production. B. HPLC profileof safracin production in wild type strain and in sacA, sacI and sacJmutants. Structure of the different molecules obtained is shown.

FIG. 7: Structure of the different molecules obtained by genedisruption. Inactivation of SacJ protein (a) yields P22B, P22A and P19molecules, whereas gene disruption of sacI (b), produces only P19compound. The sacI disruption, together with the sacJ reconstructedexpression, produces two new safracins: safracin D (possible precursorfor ET-729 hemi-synthesis) and safracin E (c).

FIG. 8: Addition of specific designed “unnatural” precursors (P3).Chemical structure of the two molecules obtained by addition of P3compound to the sacF mutant.

FIG. 9: Scheme of the gene disruption event through simplerecombination, using an homologous DNA fragment cloned into pK18:MOB (anintegrative plasmid in Pseudomonas).

DETAILED DESCRIPTION OF THE INVENTION

Non ribosomal peptide synthetases (NRPS) are enzymes responsible for thebiosynthesis of a family of compounds that include a large number ofstructurally and functionally diverse natural products. For example,peptides with biological activities provide the structural backbone forcompounds that exhibit a variety of biological activities such as,antibiotics, antiviral, antitumor, and immunosuppressive agents (Zuberet al. Biotechnology of Antibiotics 1997 (W. Strohl, ed.), 187-216Marcel dekker, Inc., N.Y; Marahiel et al. Chem. Rev. 1997, 97,2651-2673).

Although structurally diverse, most of these biologically activepeptides share a common mechanistic scheme of biosynthesis. According tothis model, peptide bond formation takes place on multienzymesdesignated peptides synthetases, on which amino acid substrates areactivated by ATP hydrolysis to the corresponding adenylate. Thisunstable intermediate is subsequently transferred to another site of themultienzymes where it is bound as a thioester to the cysteamine group ofan enzyme-bound 4′-phosphopantetheninyl (4′-PP) cofactor. At this stage,the thiol-activated substrates can undergo modifications such asepimerisation or N-methylation. Thioesterified substrate amino acids arethen integrated into the peptide product through a step-by-stepelongation by a series of transpeptidation reactions. With this templatearrangement in peptide synthetases, the modules seem to operateindependently of one another, but they act in concert to catalyse theformation of successive peptide bonds (Stachelhaus et al. Science 1995,269, 69-72; Stachelhaus et al. Chem. Biol. 1996, 3, 913-921). Thegeneral scheme for non-ribosomal peptide biosynthesis has been widelyreviewed (Marahiel et al. Chem. Rev. 1997, 97, 2651-2673; Konz andMarahiel, Chem. and Biol. 1999, 6, R39-R48; Moffit and Neilan, FEMSMicrobiol. Letters 2000, 191, 159-167).

A large number of bacterial operons and fungal genes encoding peptidesynthetases have recently been cloned, sequenced and partiallycharacterized, providing valuables insights into their moleculearchitecture (Marahiel, Chem and Biol. 1997, 4, 561-567). Differentcloning strategies were used, including probing of expression librariesby antibodies raised against peptide synthetases, complementation ofdeficient mutants, and the use of designed oligonucleotides derived fromamino acid sequences of peptide synthetase fragments.

Analysis of the primary structure of these genes revealed the presenceof distinct homologous domains of about 600 amino acids. This specificfunctional domains consist of at least six highly conserved coresequences of about three to eight amino acids in length, whose order andlocation within all known domains are very similar (Küsard and Marahiel,Peptide Research 1994, 7, 238-241). The used of degeneratedoligonucleotides derived from the conserved cores opens the possibilityof identifying and cloning peptide synthetases from genomic DNA, byusing the polymerase chain reaction (PCR) technology (Küsard andMarahiel, Peptide Research 1994, 7, 238-241; Borchert et al. FEMSMicrobiol Letters 1992, 92,175-180).

The structure of safracin suggests that this compound is synthesized bya NRPS mechanism. The cloning and expression of the non-ribosomalpeptide synthetases and the associated tailoring enzymes fromPseudomonas fluorescens A2-2 safracin cluster would allow production ofunlimited amounts of safracin. In addition, the cloned genes could beused for combinatorial creation of novel safracin analogs that couldexhibit improved properties and that could be used in the hemi-synthesisof new ecteinascidins. Moreover, cloning and expressing the safracingene cluster in heterologous systems or the combination of safracin genecluster with other NRPS genes could result in the creation of noveldrugs with improved activities.

The present invention provides, in particular, the DNA sequence encodingNRPS responsible for biosynthesis of safracin, i.e., safracinsynthetases. We have characterized a 26,705 bp region (SEQ ID NO:1) fromPseudomonas fluorescens A2-2 genome, cloned in pL30P cosmid anddemonstrated, by knockout experiments and heterologous expression, thatthis region is responsible for the safracin biosynthesis. We expressedthe pL30P cosmid in two strains of Pseudomonas sp., which do not producesafracin, and the result was a production of safracin A and B at levelsof a 22%, for P. fluorescens (CECT 378), and 2%, for P. aeruginosa (CECT110), in comparison with P. fluorescens A2-2 production. The predictedamino acids sequences of the various peptides encoded by this DNAsequence is shown in SEQ ID NO:2 through SEQ ID NO:15 respectively.

The gene cluster for safracin biosynthesis derived from P. fluorescensA2-2, is characterized by the presence of several open reading frames(ORF) that are organized in two divergent operons (FIG. 1), an eightgenes operon (sacABCDEFGH) and a two genes operon (sacIJ), preceded bywell-conserved putative promoters regions that overlap. The safracinbiosynthesis gene cluster is present in only one copy in P. fluorescensA2-2 genome.

Our results indicate that the eight genes operon would be responsiblefor the safracin skeleton biosynthesis and the two genes operon would beresponsible for the final tailoring of safracins.

In the sacABCDEFGH operon, the deduced amino acid sequences encoded bysacA, sacB and sacC strongly resemble gene products of NRPSs. Within thededuced amino acid sequences of SacA, SacB and SacC, one peptidesynthetase module was identified on each of the ORFs.

The first surprising feature of the safracin NRPS proteins is that fromthe known active sites and core regions of peptide synthetases (Konz andMarahiel, Chem. and Biol. 1999, 6, R39-R48), the first core is poorlyconserved in all three peptide synthetases, SacA, SacB and SacC (FIG.2). The other five core regions are well conserved in the three safracinNRPSs genes. The biological significance of the first core (LKAGA) isunknown, but the SGT(ST)TGxPKG (Gocht and Marahiel, J. Bacteriol. 1994,176, 2654-266; Konz and Marahiel, Chem. and Biol. 1999, 6, R39-R48), theTGD (Gocht and Marahiel, J. Bacteriol. 1994, 176, 2654-2662; Konz andMarahiel, 1999) and the KIRGxRIEL (Pavela-Vrancic et al. J. Biol. Chem1994, 269, 14962-14966; Konz and Marahiel, Chem. and Biol. 1999, 6,R39-R48) core sequences could be assigned to ATP binding and hydrolysis.The serine residue of the core sequence LGGxS could be shown to be thesite of thioester formation (D'Souza et al., J. Bacteriol. 1993, 175,3502-3510; Vollenbroich et al., FEBS Lett. 1993, 325(3), 220-4; Konz andMarahiel, Chem. and Biol. 1999, 6, R39-R48) and 4′-phosphopantetheinebinding (Stein et al. FEBS Lett. 1994, 340, 39-44; Konz and Marahiel,Chem. and Biol. 1999, 6, R39-R48). These findings, together with thefact that safracin seems to be synthesized from amino acids, supportsthe hypothesis that non-ribosomal peptide bond formation via thethiotemplate mechanism is involved in the biosynthetic pathway ofsafracin and that sacA, sacB and sacC encode the corresponding peptidesynthetases. According to this mechanism, amino acids are activated asaminoacyl-adenylates by ATP hydrolysis and subsequently covalently boundto the enzyme via carboxyl-thioester linkages. Then, in further steps,transpeptidation and peptide bond formation occurs.

Secondly, it is striking that our sequence data clearly shows that thecolinearity rule, according to which the order of the amino acid bindingmodules along the chromosome parallels the order of the amino acids inthe peptide, does not hold for the safracin synthetase system. Accordingto the sequence database homologies and safracin and saframycinstructures homologies, SacA would be responsible for the recognition andactivation of the Gly residue and SacB and SacC would be responsible forthe recognition and activation of the two L-Tyr derivatives that areincorporated into the safracin skeleton, while the putative Ala-NRPSgene would be missing in the safracin gene cluster. In a fewnonribosomal peptide synthetases gene clusters, such as in thepristamycin (Crecy-Lagard et al, J. of Bacteriol. 1997, 179(3), 705-713)and in the phosphinothricin tripeptide (Schwartz et al. Appl EnvironMicrobiol 1996, 62, 570-577) biosynthesis pathways, the first NRPS isnot juxtaposed with the second NRPS gene. In concrete, in thepristamycin biosynthetic pathway the first structural gene (snbA) andthe second structural gene (snbC) are 130 kb apart. This is not the casefor the safracin gene cluster where the results of the heterologousexpression with the pL30P cosmid clearly demonstrates that there is noNRPS gene missing since there is heterologous safracin production.

Thirdly, even though the question about the mechanism by which thedipeptide Ala-Gly is formed remains open, the presence in sacA of anextra C domain at the amino terminus of the first NRPS gene, suggeststhe possibility of a bifunctional adenylation activation activity bythis gene. We propose that the Ala would be first charged on thephosphopantetheinyl arm of SacA (FIG. 3 a* and b*) before beingtransferred to a waiting position, a condensation domain, located inN-terminal of saca (FIG. 3, c*). The Gly adenylate would then be chargedon the same phosphopantetheinyl arm (FIG. 3, d* and e*), positioned tothe elongation site, and elongation would occur (FIG. 3, f*). The arm ofthe first module would at this stage be charged with a Ala-Gly dipeptide(FIG. 3, g*). We proposed that the dipeptide would then be transferredon a waiting position in the second phosphopantetheinyl arm (FIG. 3,h*), located in SacB, to continue the synthesis of the safracintetrapeptide basic skeleton. An alternative biosynthesis mechanism couldbe the direct incorporation of a dipeptide Ala-Gly into SacA. In thiscase, the dipeptide could be originated from the activity of highlyactive peptidyl transferase ribozyme family (Sun et al, Chem. and Biol.2002, 9, 619-626) or from the activity of bacterial proteolysis.

And fourthly, although in most of the prokaryotic peptide synthetasesthe thioesterase moiety, which appears to be responsible for the releaseof the mature peptide chain from the enzyme, is fused to the C-terminalend of the last amino acid binding module (Marahiel et al. Chem. Rev.1997, 97, 2651-2673), in the case of safracin synthetases, the TE domainis missing. Probably, in the safracin synthesis after the lastelongation step, the tetrapeptide could be released by an alternativestrategy for peptide-chain termination that also occurs in thesaframycin synthesis (Pospiech et al. Microbiol. 1996, 142, 741-746).This particular termination strategy is catalysed by a reductase domainat the carboxy-terminal end of the SacC peptide synthetase whichcatalyses the reductive cleavage of the associated T-domain-tetheredacyl group, releasing a linear aldehyde.

Our cross feeding experiments indicate that the last two amino acidsincorporated into the safracin molecule are two L-Tyr derivatives calledP2 (3-hydroxy-5-methyl-O-methyltyrosine) (FIGS. 4, 5), instead of twoL-Tyr as it is proposed to occur in saframycin synthesis. First, theproducts of two genes (sacF and sacG), similar to bacterialmethyltransferases, have shown to be involved in the O-, C-methylationof L-Tyr to produce P14 (3-methyl-O-methyltyrosine), precursor of P2. Apossible mechanism could envisage that the O-methylation occurs firstand then the C-methylation of the amino acid derivative is produced.Secondly, P2, the substrate for the peptide synthetases SacB and SacC,is formed by the hydroxylation of P14 by SacD (FIGS. 4, 5).

Apart from the safracin biosynthetic genes, in the sacABCDEFGH operonthere are also found two genes, sacE and sacH, involved in an unknownfunction and in the safracin resistance mechanism, respectively. We havedemonstrated that sacH gene codes for a protein that when isheterologous expressed, in different Pseudomonas strains, a highlyincrease of the safracin B resistance is produced. SacH is a putativetransmembrane protein, that transforms the C₂₁—OH group of safracin Binto a C₂₁—H group, to produce safracin A, a compound with lessantibiotic and antitumoral activity. Finally, even though still isunknown about the putative function of SacE, homologous of this genehave been found close to various secondary metabolites biosynthetic geneclusters in some microorganisms genomes, suggesting a conserved functionof this genes in secondary metabolite formation or regulation.

In the sacIJ operon, the deduced amino acid sequences encoded by sacIand sacJ strongly resemble gene products of methyltransferase andhydroxylase/monoxygenase, respectively. Our data reveals that SacI isthe enzyme responsible for the N-methylation present in the safracinstructure, and that SacJ is the protein which makes an additionalhydroxylation on one of the L-Tyr derivative incorporated into thetetrapeptide to produce the quinone structure present in all safracinmolecules. N-Methylation is one of the modifications of nonribosomallysynthesized peptides that significantly contributes to their biologicalactivity. Except for saframycin (Pospiech et al. Microbiol. 1996, 142,741-746), that is produced by bacteria and is N-methylated, all theN-methylated nonribosomal peptides known are produced by fungi oractinomycetes and, in most of the cases, the responsible for theN-methylation is a domain which reside in the nonribosomal peptidesynthetase. TABLE I Summary of safracin biosynthetic and resistancegenes identified in cosmid pL30P. Pro- ORF tein Position Amino Molecularname name Proposed function start-stop bp acids weight sacA SacA Peptidesynthetase 3052-6063 1004 110.4 sacB SacB Peptide synthetase 6068-92681063 117.5 sacC SacC Peptide synthetase  9275-13570 1432 157.3 sacD SacDL-Tyr derivative 13602-14651 350 39.2 hidroxylase sacE SacE Unknown14719-14901 61 6.7 sacF SacF L-Tyr derivative 14962-16026 355 39.8methylase sacG SacG L-Tyr O-methylase 16115-17155 347 38.3 sacH SacHResistance protein 17244-17783 180 19.6 sacI SacI methyl-transferase2513-1854 220 24.2 sacJ SacJ monooxygenase 1861-355  509 55.3

The safracin putative synthetic pathway, with indications of thespecific amino acid substrates used for each condensation reaction andthe various post-condensation activities, is shown in FIG. 5.

To further evaluate the role of safracin biosynthetic genes, weconstructed knock out mutants of each of the genes of the safracincluster (FIG. 6). The disruption of the NRPSs genes (sacA, sacB andsacC) as well as sacD, sacF and sacG, resulted in safracin and P2 nonproducing mutants. Our results indicate that the genes from sacA to sacHare part of the same genetic operon. As a consequence of the sacI andsacJ gene disruptions three new molecules were originated, P19B, P22Aand P22B (FIG. 6).

The production of P22A and P22B (FIG. 7 a*) by sacJ mutant demonstratedthat the role of the SacJ protein is to produce the additionalhydroxylation of the left L-Tyr derivatives amino acid of the safracin,the one involved in the quinone ring. The production of P19B (FIG. 7 b*)by sacI mutant, a safracin like molecule where the N-methylation and thequinone ring are missing, confirms that SacI is the N-methyltransferaseenzyme and suggests that sacIJ is a transcriptional operon. Theproduction of P19B also by sacJ mutant (FIG. 7 a*) suggests thatprobably the N-methylation occurs after the quinone ring has beenformed. Even though these new structures have no interestingantimicrobial activity on B. subtilis or no high citotoxic activity oncancer cells, they can serve as interesting new precursors for thehemisynthesis of new active molecules. As far as structure activity isconcerned, the observation that P19B, P22A and P22B appear to loosetheir activity, suggests that the lost of the quinone ring from thesafracin structure is directly related with the lost of activity of thesafracin family molecules.

The disruption of sacI gene with the reconstitution of the sacJ geneexpression resulted in the production of two new safracins. The twoantibiotics produced, at levels of production as high as the levels ofsafracin A/safracin B production in the wild type strain, have beennamed as safracin D and safracin E (FIG. 7 c*).

The safracin D and safracin E are safracin B and safracin A likemolecules, respectively, where the N-methylation is missing. Both,safracin D and safracin E have been shown to possess the sameantibacterial and antitumoral activities as safracin B and safracin A,respectively. Apart from its high activities properties, antibacterialand antitumoral, safracin D could be used in the hemi-synthesis of theecteinascidin ET-729, a potent antitumoral agent, as well as in thehemi-synthesis of new ecteinascidins.

A question arises concerning the role of the aminopeptidase-like proteincoded by a gene located at 3′site of the safracin operon. Theinsertional inactivation of orf1 (PM-S1-14) showed no effect on safracinA/safracin B production. Because of its functionality properties itremains unclear if this protein could play some role in the safracinmetabolism. The other genes present in the pL30P cosmid (orf2 to orf4)will have to be studied in more detail.

Another aspect of the invention is that provides the tools necessary forthe production of new specific designed “unnatural” molecules. Theaddition of a specific modified P2 derivative precursor named P3, a3-hydroxy-5-methyl-O-methyltyrosine, to the sacE mutant yields two“unnatural” safracins that incorporated this specific modifiedprecursor, safracin A(OEt) and safracin B(OEt) (FIG. 8).

The two new safracins are potent antibiotic and antitumoral compounds.The biological activities of safracin A(OEt) and Safracin B(OEt) are aspotent as the activities of safracin A and safracin B, respectively.These new safracins could be the source for new potent antitumoralagents, as well as a source of molecules for the hemi-synthesis of newecteinascidins.

In addition, the genes involved in safracin synthesis could be combinedwith other non ribosomal peptide synthetases genes to result in thecreation of novel “unnatural” drugs and analogs with improvedactivities.

EXAMPLES Example 1 Extraction of Nucleic Acid Molecules from Pseudomonasfluorescens A2-2

Bacterial Strains

Strains of Pseudomonas sp. were grown at 27° C. in Luria-Bertani (LB)broth (Ausubel et al. 1995, J. Wiley and Sons, New York, N.Y). E. colistrains were grown at 37° C. in LB medium. Antibiotics were used at thefollowing concentrations: ampicillin (50 μg/ml), tetracycline (20 μg/ml)and kanamycin (50 μg/ml). TABLE II Strains used in this invention. CodeGenotype PM-S1-001 P. fluorescens A2-2 wild type PM-S1-002 sacA-PM-S1-003 sacB- PM-S1-004 sacC- PM-S1-005 sacJ- PM-S1-006 sacI-PM-S1-007 sacI- with sacJ expression reconstitution PM-S1-008 sacF-PM-S1-009 sacG- PM-S1-010 sacD- PM-S1-014 orf1- PM-S1-015 A2-2 + pLAFR3PM-S1-016 A2-2 + pL30p PM-19-001 P. fluorescens CECT378 + pLAFR3PM-19-002 P. fluorescens CECT378 + pL30p PM-19-003 P. fluorescensCECT378 + pBBR1-MCS2 PM-19-004 P. fluorescens CECT378 + pB5H83 PM-19-005P. fluorescens CECT378 + pB7983 PM-19-006 P. fluorescens CECT378 +pBHPT3 PM-16-001 P. aeruginosa CECT110 + pLAFR3 PM-16-002 P. aeruginosaCECT110 + pL30p PM-17-003 P. putida ATCC12633 + pBBR1-MCS2 PM-17-004 P.putida ATCC12633 + pB5H83 PM-17-005 P. putida ATCC12633 + pB7983PM-18-003 P. stutzeri ATCC17588 + pBBR1-MCS2 PM-18-004 P. stutzeriATCC17588 + pB5H83 PM-18-005 P. stutzeri ATCC17588 + pB7983DNA Manipulation

Unless otherwise noted, standard molecular biology techniques for invitro DNA manipulations and cloning were used (Sambrook et al. 1989,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory).

DNA Extraction

Total DNA from Pseudomonas fluorescens A2-2 cultures was prepared asreported (Sambrook et al. 1989, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory).

Computer Analysis

Sequence data were compiled and analysed using DNA-Star softwarepackage.

Example 2 Identification of NRPS Genes Responsible for SafracinProduction in Pseudomonas fluorescens A2-2

Primer Design

Marahiel et al. (Marahiel et al. Chem. Rev. 1997, 97, 2651-2673)previously reported highly conserved core motifs of the catalyticdomains of cyclic and branched peptide synthetases. Based on multiplesequence alignments of several reported peptide synthetases theconserved regions A2, A3, A5, A6, A7 and A8 of adenylation and T ofthiolation modules were targeted for the degenerate primer design(Turgay and Marahiel, Peptide Res. 1994, 7, 238-241). The wobblepositions were designed in respect to codon preferences within theselected modules and the expected high G/C content of Pseudomonas sp.All oligonucleotides were obtained from ISOGEN (Bioscience BV). A PCRfragment was obtained when degenerate oligonucleotides derived from theYGPTE (A5 core) and LGGXS (T core) sequences were used. Theseoligonucleotides were denoted PS34-YG and PS6-FF, respectively. TABLEIII PCR primers designed for this study. Primer designation andorientation Sequence Length PS34-YG (forward) 5′-TAYGGNCCNACNGA-3′14-mer PS6-FF (reverse) 5′-TSNCCNCCNADNTCRAARAA-3′ 20-merPCR Conditions for Amplification of DNA from P. fluorescens A2-2

A fragment internal to nonribosomal peptide synthetases (NRPS) wasamplified using PS-34-YG and PS6-FF oligonucleotides and P. fluorescensA2-2 chromosomal DNA as template. Reaction buffer and Taq polymerasefrom Promega were used. The cycling profile performed in a Personalthermocycler (Eppendorf) consists on: 30 cycles of 1 min at 95° C., 1min at 50° C., 2 min at 72° C. PCR products were on the expected size(750 bp aprox.) based on the location of the primers within the NRPSdomains of other synthetase genes.

DNA Cloning

PCR amplification fragments were cloned into pGEM-Teasy vector accordingto the manufacturer (Qiagen, Inc., Valencia, Calif.). In this way,cloned fragments are flanked by two EcoRI restriction sites, in order tofacilitate subsequent subclonig in other plasmids (see below). SinceNRPSs enzymes are modular, clones from the degenerated PCR primersrepresents a pool of fragments from different domains.

DNA Sequencing

All sequencing was performed using primers directed against the cloningvector, with an ABI Automated sequencer (Perkin-Elmer). Cloned DNAsequences were identified using the BLAST server of the National Centerfor Biotechnology Information accessed over the Internet (Altschul etal., Nucleic Acids Res. 1997, 25, 3389-3521). All of the sequences havesignature regions for NRPSs and show high similarity in BLAST searchesto bacterial NRPS showing that they are in fact of peptide origin.Moreover, a probable domain similarity search was performed using thePROSITE (European Molecular Biology Laboratory, Heidelberg, Germany) webserver.

Gene Disruption of Pseudomonas fluorescens A2-2

In order to analyse the function of the genes cloned, these genes weredisrupted through homologous recombination (FIG. 9). For this purpose,recombinant plasmids (pG-PS derivatives) harbouring the NRPS genefragment were digested with EcoRI restriction enzyme. The resultingfragments belonging to the gene to be mutated were cloned into thepK18mob mobilizable plasmid (Schäfer et al. Gene 1994, 145, 69-73), achromosomal integrative plasmid able to replicate in E. coli but not inPseudomonas strains. Recombinant plasmids were introduced first in E.coli S17-λPIR strain by transformation and then in P. fluorescens A2-2through biparental conjugation (Herrero et al, J Bacteriol 1990, 172,6557-6567). Different dilutions of the conjugation were plated onto LBsolid medium containing ampicillin plus kanamycin and incubatedovernight at 27° C. Kanamycin-resistant transconjugants, containingplasmids integrated into the genome via homologous recombination, wereselected.

Biological Assay (biotest) for Safracin Production

Strains P. fluorescens A2-2 and its derivatives were incubated in 50 mlbaffled erlenmeyer flasks containing fermentation medium with thecorresponding antibiotics. Initially, SA3 fermentation medium was used(Ikeda Y. J. Ferment. Technol. 1985, 63, 283-286). In order to increasethe productivity of the fermentation process statistical-mathematicalmethods like Plackett-Burman designed was used to select nutrients andresponse surface optimisation techniques were tested (Hendrix C.Chemtech 1980, 10, 488-497) in order to determine the optimum level ofeach key independent variable. Experiments to improve the cultureconditions like incubation temperature and agitation have also beendone. Finally a highly safracin B producer medium named 16B (152 g/l ofmannitol, 35 g/l of G20-25 yeast, 26 g/l of CaCO₃, 14 g/l of ammoniumsulphate, 0.18 g/l of ferric chloride, pH 6.5) was selected.

The safracin production was assay testing the capacity of inhibition aBacillus subtilis solid culture by 10 μl of the supernatant of a 3 daysPseudomonas sp. culture incubated at 27° C. (Alijah et al. ApplMicrobiol Biotechnol 1991, 34, 749-755). P. fluorescens A2-2 culturesproduce inhibition zones of 10-14 mm diameter while non-producingmutants did not inhibit B. subtilis growth. Three isolated clones hadthe safracin biosynthetic pathway affected. In order to confirm theresults, HPLC analysis of safracin production was performed.

HPLC Analysis of Safracin Production.

The supernatant was analysed by using HPLC Symmetry C-18. 300 Å, 5 μm,250×4.6 mm column (Waters) with guard-column (Symmetry C-18, 5 μm 3.9×20mm, Waters). An ammonium acetate buffer (10 mM, 1% Diethanolamine, pH4.0)-acetonitrile gradient was the mobile phase. Safracin was detectedby absorption at 268 nm. In FIG. 6, HPLC profile of safracin andsafracin precursors produce by P. fluorescens A2-2 strain and differentsafracin-like structures produced by P. fluorescens mutants are shown.

Example 3 Cloning and Sequence Analysis of Safracin Cluster

Inverse PCR and Phage Library Hybridisation

Southern hybridisation on mutant chromosomal DNAs verified the correctgene disruption and demonstrated that the peptide synthethase fragmentcloned into pK18mob plasmid was essential for the production ofsafracin. Analysis of the non safracin producers mutants obtaineddemonstrated that all of them had a gene disruption into the same gene,sacA.

Inverse PCR from genomic DNA and screening of a phage library of P.fluorescens A2-2 genomic DNA revealed the presence of additional genesflanking sacA gene, probably involved in safracin biosynthesis.

The GenBank accession number for the nucleotide sequence data of the P.fluorescens A2-2 safracin biosynthetic cluster is AY061859.

Cosmid Library Construction and Heterologous Expression

To determine whether safracin cluster was able to confer safracinbiosynthetic capability to a non producer strain, it was cloned into awide range cosmid vector (pLAFR3, Staskawicz B. et al. J Bacteriol 1987,169, 5789-5794) and conjugated to a different Pseudomonas sp collectionstrains.

To obtain a clone containing the whole cluster, a cosmid library wasconstructed and screened. For this purpose, chromosomal DNA waspartially digested with the restriction enzyme PstI, the fragments weredephosphorylated and ligated into the PstI site of cosmid vector pLAFR3.The cosmids were packaged with Gigapack III gold packaging extracts(Stratagene) as manufacturer's recommendations. Infected cells of strainXL1-Blue were plated on LB-agar supplemented with 50 μg/ml oftetracycline. Positives clones were selected using colony hybridizationwith a DIG-labeled DNA fragment belonging to the 3′-end of the safracincluster. In order to ensure the cloning of the whole cluster, a newcolony hybridization with a 5′-end DNA fragment was done. Only cosmidpL30p showed multiple hybridizations with DNA probes. To confirm theaccurate cloning, PCR amplification and DNA-sequencing with DNAoligonucleotides belonging to the safracin sequence were carried out.The size of the insert of pL30P was 26,705 bp. The pL30p clone DNA wastransformed into E. coli S17λPIR and the resulting strain wereconjugated with the heterologous Pseudomonas sp. strains. The pL30pcosmid was introduced into P. fluorescens CECT378 and P. aeruginosaCECT110 by biparental conjugation as described above. Once a cloneencoding the whole cluster was identified, it was determined whether thecandidate was capable of producing safracin. Safracin production in theconjugated strains was assessed by HPLC analysis and biological assay ofbroth cultures supernatants as previously described.

The strain P. fluorescens CECT378 expressing the pL30p cosmid(PM-19-002) was able to produce safracin in considerable amounts,whereas safracin production in P. aeruginosa CECT110 strain expressingpL30P (PM-16-002) was 10 times less than the CECT378. Safracinproduction in these strains was about 22% and 2% of the total productionin comparison with the natural producer strain.

Genes Involved in the Formation of Safracin. SEQUENCE Analysis ofsacABCDEFGH and sacIJ Operons

Computer analyses of the DNA sequence of pL30P revealed 14 ORFs (FIG.1). A potential ribosome binding site precedes each of the ATG startcodons.

In the sacABCDEFGH operon, three very large ORFs, sacA, sacB and sacC(positions 3052 to 6063, 6080 to 9268 and 9275 to 13570 of the P.fluorescens A2-2 safracin sequence SEQ ID NO:1, respectively) can beread in the same direction and encode the putative safracin NRPSs: SacA(1004 amino acids, M_(r) 110452), SacB (1063 amino acids, M_(r) 117539)and SacC (1432 amino acids, M_(r) 157331). The three NRPSs genes containthe domains resembling amino acid activating domains of known peptidesynthetases. Specifically, the amino acid activating domains from theseNRPS genes are very similar to three of the four amino acid activatingdomains (Gly, Tyr and Tyr) found in the Myxococcus xanthus saframycinNRPSs (Pospiech et al. Microbiology 1995, 141, 1793-803; Pospiech et al.Microbiol. 1996, 142, 741-746). In particular, SacA (SEQ ID NO:2) shows33% identity with saframycin Mx1 synthetase B protein (SafB) from M.xanthus (NCBI accession number U24657), whereas SacB (SEQ ID NO:3) andSacC (SEQ ID NO:4) share, respectively, 39% and 41% identity withsaframycin Mx1 synthetase A (SafA) from M. xanthus (NCBI accessionnumber U24657). The FIG. 2 shows a comparison among SacA, SacB y SacCand the different amino acid activating domains of saframycin NRPS.

Downstream sacC five small ORFs reading in the same direction as theNRPSs genes exist (FIG. 1). The first one, sacD (position 13602 to 14651of P. fluorescens A2-2 safracin sequence), encodes a putative protein,SacD (350 amino acids, M_(r) 39187; SEQ ID NO:5), with no similaritiesin the GeneBank DB. The next one, sacE (position 14719 to 14901 of P.fluorescens A2-2 safracin sequence), encodes a small putative proteincalled SacE (61 amino acids, M_(r) 6729; (SEQ ID NO:6)), which showssome similarity with proteins of unknown function in the databases (ORF1from Streptomyces viridochromogenes (NCBI accession number Y17268; 44%identity) and MbtH from Mycobacterium tuberculosis (NCBI accessionnumber Z95208; 36% identity). The third ORF, sacF (position 14962 to16026 of P. fluorescens A2-2 safracin sequence), encodes a 355-residueprotein with a molecular weigh calculated of 39,834 (SEQ ID NO:7). Thisprotein most closely resembles hydroxyneurosporene methyltransferase(CrtF) from Chloroflexus aurantiacus (NCBI accession number AF288602;25% identity). The nucleotide sequence of the fourth ORF, sacG (position16115 to 17155 of P. fluorescens A2-2 safracin sequence), predicted agene product of 347 amino acids having a molecular mass of 38,22 kDa(SEQ ID NO:8). The protein, called SacG, is similar to bacterialO-methyltransferases, including O-dimethylpuromycin-O-methyltransferase(DmpM) from Streptomyces anulatus (NCBI accession number P42712; 31%identity). A computer search also shows that this protein contains thethree sequence motifs found in diverse S-adenosylmethionine-dependentmethytransferases (Kagan and Clarke, Arch Biochem. Biophys. 1994, 310,417-427). The fifth gene, sacH (position 17244 to 17783 of P.fluorescens A2-2 safracin sequence), encodes a putative protein SacH(180 amino acids, M_(r) 19632; (SEQ ID NO:9). A computer search forsimilarities, between the deduced amino acid sequence of SacH and otherprotein sequences, revealed identity with some conserved hypotheticalproteins of unknown function, which contains a well conservedtransmembrane motif and a dihydrofolate reductase-like active site(Conserved hypothetical protein from Pseudomonas aeruginosa PAO1, NCBIaccession number P3469; 35% identity).

Upstream sacABCDEFGH operon, reading in opposite sense, a two genesoperon, sacIJ, is located. The sacI gene (position 2513 to 1854) encodesa 220-amino acids protein (M_(r) 24219; (SEQ ID NO: 10) that mostclosely resembles ubiquinone/manequinone methyltrasnferase fromThermotoga maritime (NCBI accession number AE001745; 32% identity). ThesacJ gene (position 1861 to 335) encodes a 509-amino acid protein (SEQID NO:11), with a molecular mass of 55341 Da, similar to bacterialmonooxygenases/hydroxylases, including putative monooxygenase fromBacillus subtilis (NCBI accession number Y14081; 33% identity) andStreptomyces coelicolor (NCBI accession number AL109972; 29% identity).

SacABCDEFGH and sacIJ operons are transcribed divergently and areseparated by 450 bp approximately. Both operons are flanked by residualtransposase fragments.

Related Safracin Cluster Genes

A putative ORF (orf1; position 18322 to 19365 of P. fluorescens A2-2safracin sequence) located at the 3′-end of the safracin sequence hasbeen found (FIG. 1). ORF1 protein (SEQ ID NO:12) shows similarity withaminopeptidases from the Gene Bank DataBase (peptidase M20/M25/M40family from Caulobacter crescentus CB15; NCBI accession number NP422131;30% identity). Using the strategy described in Example 2, the genedisruption of orf1 do not affect safracin production in P. fluorescensA2-2.

At the 3′-end of the safracin sequence cloned in pL30p cosmid, threeputative ORFs (orf2, orf3 and orf4), were found. Reading in oppositedirection than sacABCDEFGH operon, orf2 gene (position 22885 to 21169 ofSEQ ID NO:1) codes for a protein, ORF2 (SEQ ID NO:13), with similaritiesto Aquifex aeolicus HoxX sensor protein (NCBI accession numberNC000918.1; 35% identity), whereas orf3 gene (position 23730 to 23041 ofSEQ ID NO:1) codes for ORF3 protein (SEQ ID NO:14) which shares 44%identity with a glycosil transferase related protein from Xanthomonasaxonopodis pv. Citri str. 306 (NCBI accession number NP642442).

The third gene is located at the 3′-end of SEQ ID NO:1 (position 25037to 26095). This gene, named orf4 (position 2513 to 1854), encodes aprotein, ORF4 (SEQ ID NO:15), that most closely resembles to ahypothetical isochorismatase family protein YcdL from Escherichia coli.(NCBI accession number P75897; 32% identity).

Presumably, these three genes would not be involve in the safracinbiosynthetic pathway, however, future gene disruption of these geneswill confirm this assumption.

The different DNA sequences found are listed at the end of thedescription.

Example 4 Functional Analysis of the Safracin Loci and Search forPossible Precursors

Since the pathway for synthesis of safracin in P. fluorescens A2-2 is atpresent unknown, the inactivation of each of the genes described inExample 3 would permit fundamental studies on the mechanism of safracinbiosynthesis in this strain.

In order to analyze the functionality of each particular protein in thesafracin production pathway, disruption of each particular gene of thecluster, but sacE, was performed. All of the genetic mutants wereobtained following the disruption strategy previously described.

FIG. 6 is a summary of the different mutants constructed in thisinvention as well as a summary of the compounds produced by the mutantsas a consequence of the gene disruption. In the wild type strain bothsafracin A and B and other compounds, P2 and P14, were clearly detectedby HPLC (see FIG. 6,WT). The gene disruption of the saca (PM-S1-002),sacB (PM-S1-003), sacC (PM-S1-004), sacD (PM-S1-010), sacF (PM-S1-008),and sacG (PM-S1-009), genes generated mutants that were unable toproduce neither safracin A and safracin B, nor the precursor compoundswith retention times beneath 15 min, P2 and P14 respectively. Thestructure elucidation of P14 and P2 revealed that P14 is a3-methyl-O-methyl tyrosine, where as P2 is a 3-hydroxy-5-methyl-O-methyltyrosine. Because of the small size of the sacE gene, the sacE⁻ mutantwas not possible to be obtained by gene disruption, but deletion of thisgene is in process. The overexpression of SacE protein, in trans, had noeffect on safracin B/A production. The sacI⁻ mutants (PM-S1-006)produced P2, P14 and significant amount of a compound called P19B (FIG.6; FIG. 7 b*). Structure elucidation of P19B revealed that this compoundis a safracin-like molecule in which the N-Met and one of the OH fromthe quinone ring are missing. In the sacJ⁻ mutants (PM-S1-005), P2, P14,P19B and two new compounds called P22A and P22B were obtained (FIG. 6;FIG. 7 a*). Structure elucidation of P22A and P22B revealed that theyare safracin A and safracin B like molecules, respectively, without oneof the —OH group from the quinone ring. The biological assay of thesacI⁻ and the sacJ⁻ mutants extracts revealed very low activity againstBacillus subtilis.

The disruption of sacI gene with the reconstitution of the sacJ geneexpression resulted in a new safracins producer mutant, PM-S1-007. Thetwo antibiotics produced, at levels of production as high as the levelsof safracin A and safracin B in the wild type strain, have been named assafracin D and safracin E (FIG. 7 c*). The safracin D and safracin E aresafracin B and safracin A like molecules, respectively, where theN-methylation is missing.

These results strongly suggest that i) sacA, sacB and sacC genes encodefor the safracin NRPSs; ii) sacD, sacF and sacG genes are responsiblefor the transformation of L-Tyr into the L-Tyr derivative P2 and iii)sacI and sacJ are responsible for the tailoring modifications thatconvert P19 and P22 into safracin.Characterization of Natural Precursors:

Strain:Pseudomonas fluorescens A2-2 (wild type) (PM-S1-001)Fermentation Conditions:

Seed medium YMP3 containing 1% glucose; 0.25% beef extract; 0.5%bacto-peptone; 0.25% NaCl; 0,8% CaCO3 was inoculated with 0.1% of afrozen vegetative stock of the microorganism, and incubated on a rotaryshaker (250 rpm) at 27° C. After 30 h of incubation, the 2% (v/v) seedculture was transferred into 2000 ml Erlenmeyer flasks containing 250 mlof the M-16B production medium, composed of 15.2% mannitol; 3.5% Driedbrewer's yeast; 1.4% (NH₄)₂ SO₄; 0.001%; FeCl₃; 2.6% CO₃Ca. Thetemperature of the incubation was 27° C. from the inoculation till 40hours and then, 24° C. to final process (71 hours). The pH was notcontrolled. The agitation of the rotatory shaker was 220 rpm with 5 cmeccentricity.

Isolation:

After 71 hours of incubation, 2 Erlenmeyer flasks were pooled and the500 ml of fermentation broth was clarified by 7.500 rpm centrifugationduring 15 minutes. 50 grams of the resin XAD-16 (Amberlite) were addedto the supernatant and mixed during 30 minutes at room temperature.Then, the resin was recovered from the clarified broth by filtration.The resin was washed twice with distilled water and extracted with 250ml of isopropanol (2-PrOH). The alcohol extract was dried under highvacuum till obtention of 500 mg crude extract. This crude was dissolvedin methanol and purified by chromatographic column using Sephadex LH-20and methanol as mobile phase. The P-14 compound was eluted and dried asa 15 mg yellowish solid. The purity was tested by analytical HPLC and ¹HNMR.

P-14 was also isolated in a similar way from cultures of the sacJ⁻mutant (PM-S1-005), using semipreparative HPLC as the last step in thepurification process.

Biological Activities:

NO ACTIVE

Spectroscopic Data:

ESMS m/z 254 (C₁₁H₁₄NO₃Na₂ ⁺), 232 (C₁₁H₁₅NO₃Na⁺), 210 (M+H⁺). ¹H RMN(300 MHz, CD₃OD): 7.07 (d, J=8.1 Hz, H-9), 7.06 (s, H-5), 6.84 (d, J=8.1Hz, H-8), 3.79 (s, H-11), 3.72 (dd, J=8.7, 3.9 Hz, H-2), 3.20 (dd,J=14.4, 3.9 Hz, H-3a), 2.91 (dd, J=14.4, 8.9 Hz, H-3b), 2.16 (s, H-10).¹³C RMN (75 MHz, CD₃OD): 174.1 (C-1), 158.6 (C-7), 132.5 (C-5), 128.9(C-9), 128.5 (C-4), 128.0 (C-6), 111.4 (C-8), 57.6 (C-2), 55.8 (C-11),37.4 (C-3), 16.3 (C-10)

Strain:Pseudomonas fluorescens A2-2 (wild type) (PM-S1-001)Fermentation Conditions:

The same process than P-14

Isolation:

Similar procedure as the P-14, except in the Sephadex chromatography,where the fractions containing P-2 have eluted later. A semi-preparativeHPLC step (Symmetry Prep C-18 column, 7.8×150 mm, AcONH₄ 10 mMpH=3/CH₃CN 95:5 held for 5 min and then gradient from 5 to 6.8% of CH₃CNin 3 min) has been necessary to purify the P-2. Also this compound hasbeen isolated from the fermentation broth of the Pseudomonas putidaATCC12633+pB5H83 (PM-17-004) as result of heterologous expression.

Biological Activities:

NO ACTIVE

Spectroscopic Data:

ESMS m/z 226 [M+H]⁺; ¹H RMN (CD₃OD, 300 MHz): 6.65 (d, J=1.8 Hz, H-5),6.59 (d, J=1.8 Hz, H-9), 3.72 (s, H-11), 3.71 (dd, J=9.0, 4.2 Hz, H-2),3.16 (dd, J=14.4, 4.2 Hz, H-3a), 2.83 (dd, J=14.4, 9.0 Hz, H-3b), 2.22(s, H-10); ¹³C RMN (DMSO, 75 MHz): 170.88 (s, C-1), 150.025 (s, C-7),144.56 (s, C-8), 132.28 (s, C-4), 130.36 (s, C-6), 121.73 (d, C-5),115.55 (d, C-9), 59.06 (q, 7-OMe), 55.40 (d, C-2), 36.21 (t, C-3), 15.86(q, 6-Me).Characterization of Safracins like Compounds Obtained by Knock Out

Strain:sac J⁻ mutant from P. fluorescens A2-2 (PM-S1-005)Fermentation conditions:

50 liters of the SAM-7 medium (50 l) composed of dextrose (3.2%),mannitol (9.6%), dry brewer's yeast (2%), ammonium sulphate (1.4%),potassium secondary phosphate (0.03%), potassium chloride (0.8%), Iron(III) chloride 6-hydrtate (0.001%), L-tyrosine (0.1%), calcium carbonate(0.8%), poly-(propylene glycol) 2000 (0.05%) and antifoam ASSAF 1000(0.2%) was poured into a jar-fermentor (Bioengineering LP-351) with 75 ltotal capacity and, after sterilization, sterile antibiotics(amplicillin 0.05 g/l and kanamycin 0.05 g/l) were added. Then, it wasinoculated with seed culture (2%) of the mutant strain PM-S1-005. Thefermentation was carried out during 71 h. under aerated and agitatedconditions (1.0 l/l/min and 500 rpm). The temperature was controlledfrom 27° C. (from the inoculation till 24 hours) to 25° C. (from 24 h tofinal process). The pH was controlled at pH 6.0 by automatic feeding ofdiluted sulphuric acid from 22 hours to final process.

Isolation

The whole broth was clarified (Sharples centrifuge). The pH of theclarified broth was adjusted to pH 9.0 by addition of NaOH 10% andextracted with 25 litres of ethyl acetate. After 20′ mixing, the twophases were separated. The organic phase was frozen overnight and then,filtered for removing ice and evaporated to a greasy dark green extract(65.8 g). This extract was mixed with 500 ml hexane (250 ml two times)and filtered for removing hexane soluble impurities. The remainingsolid, after drying, gave a 27.4 g of a dry green-beige extract.

This new extract was dissolved in methanol and purified by a SephadexLH-20 chromatography (using methanol as mobile solvent) and thesafracins-like compounds were eluted in the central fractions (Analyzedon TLC conditions: Silica normal phase, mobile phase: EtOAc:MeOH 5:3.Aprox. Rf valor: 0.3 for P-22B, 0.25 P-22A and 0.1 for P-19).

The pooled fractions, (7,6 g) containing the three safracin-likecompound were purified by a Silica column using a mixture of EtOAc:MeOHfrom 50:1 to 0:1. and other chromatographic system (isocraticCHCl₃:MeOH:H₂O:AcOH 50:45:5:0.1). Compounds P22-A, P22-B and P19-B werepurified by reversed-phase HPLC (SymmetryPrep C-18 column 150×7.8 mm, 4mL/min, mobile phase: 5 min MeOH:H₂O (0.02% TFA) 5:95 and gradient fromMeOH:H₂O (0.02% TFA) 5:95 to MeOH 100% in 30 min).

Biological Activities of Safracin P-22B Cells Lines (Mol/L) PrimaryProstate Ovary Breast Melanoma Endothelio Screening DU-145

SK-OV-3 IGROV IGROV-ET SK-BR3 SK-MEL-28 HYECI PM- GI50 4.58E−06 3.08E−078.49E−07 3.02E−06 8.24E−07 5.20E−07 TGI 8.62E−06 6.08E−07 2.30E−067.01E−06 2.28E−06 9.99E−07 23-0

LC50 1.62E−05 1.20E−06 1.21E−05 1.65E−05 8.85E−06 2.01E−06 Primary NSCLLeukemia Pancreas Colon Cervix Screening A549 K-562 PANCI HT29 LOVOLOVO-DOX HELA HELA-AFL PM- GI50 4.71E−06 1.13E−07 4.77E−06 1.01E−062.54E−06 6.95E−06 7.61E−07 4.65E−07 TGI 8.83E−06 4.67E−07 1.17E−052.75E−06 6.84E−06 1.90E−05 1.83E−06 9.32E−07 23-O

LC50 1.66E−05 1.84E−06 >1.90E−05   1.86E−05 1.84E−05 >1.90E−05  7.42E−06 1.86E−06Antimicrobial activity: On solid mediumBacillus subtilis. 10 μg/disk (6 mm diameter): 10 mm inhibition zoneSpectroscopic Data:HRFABMS m/z 509.275351 [M-H₂O+H]⁺ (calcd for C₂₈H₃₇N₄O₅ 509.276396 Δ1.0mmu); LRFABMS using m-NBA as matrix m/z (rel intensity) 509 [M-H₂O+H]⁺(5), 460 (2.7), 391 (3).

¹H NMR (CD₃OD, 500 MHz): 6.70 (s, H-15), 6.52 (s, H-5), 4.72 (bs, H-11),4.66 (d, J=2.0 Hz, H-21), 4.62 (dd, J=8.4, 3.7 Hz, H-1), 3.98 (bd, J=7.6Hz, H-13), 3.74 (s, 7-OMe), 3.71 (s, 17-OMe), 3.63 (m, overlappedsignal, H-25), 3.62 (m, overlapped signal, H-3), 3.30 (m, H-22a), 3.29(m, H-14a), 3.18 (d, J=18.6 Hz, H-14b), 2.90 (m, H-4a), 2.88 (m, H-22b),2.76 (s, 12-NMe), 2.30 (s, 16-Me), 2.22 (m, H-4b), 1.16 (d, J=7.4 Hz,H-26);

¹³C NMR (CD₃OD, 125 MHz): 170.75 (s, C-24), 149.24 (s, C-18), 147.54 (s,C-8), 145.95 (s, C-7), 145.82 (s, C17), 133.93 (s, C-16), 132.31 (s,C-9), 131.30 (s, C-6), 128.95 (s, C-20), 121.93 (d, C-15), 121.76 (d,C-5), 121.44 (s, C-10), 112.45 (s, C-19), 92.87 (d, C-21), 60.86 (q,7-OMe), 60.76 (q, 17-OMe), 59.39 (d, C-11), 57.96 (d, C-13), 55.51 (d,C-1), 54.29 (d, C-3), 50.08 (d, C-25), 45.55 (t, C-22), 40.43 (q,12-NMe), 32.56 (t, C-4), 25.84 (t, C-14), 17.20 (q, C-26), 16.00 (q,16-Me), 15.81 (q, 6-Me).

Strain:The same as for P-22BFermentation Conditions:The same as for P-22BIsolation:The same as for P-22BBiological Activities of Safracin P-22A

Antitumor Activities Cells Lines (Mol/L) Prostate Ovary Breast MelanomaEndothelio Primary Screening DU-145

SK-OV-3 IGROV IGROV-ET SK-BR3 SK-MEL-28 HYECI Safracin P-22AGI50 >1.96E−05 4.19E−06 7.74E−06 1.30E−05 1.27E−05 5.93E−06TGI >1.96E−05 9.26E−06 1.96E−05 >1.96E−05   >1.96E−05 1.33E−05LC50 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 NSCLLeukemia Pancreas Colon Cervix Primary Screening A549 K-562 PANCI HT29LOVO LOVO-DOX HELA HELA-AFL Safracin P-22A GI50 >1.96E−053.15E−06 >1.96E−05   1.26E−05 >1.96E−05 >1.96E−05 8.75E−06 7.66E−06TGI >1.96E−057.93E−06 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05LC50 >1.96E−051.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05 >1.96E−05Antimicrobial activity: On solid mediumBacillus subtilis. 10 μg/disk (6 mm diameter): NO ACTIVESpectroscopic data:

HRFABMS m/z 511.290345 [M+H]⁺ (calcd for C₂₈H₃₉N₄O₅ 511.292046 A 1.7mmu); LRFABMS using m-NBA as matrix m/z (rel intensity) 511 [M+H]⁺ (61),409 (25), 391 (4); ¹H NMR (CD₃OD, 500 MHz): 6.68 (s, H-15), 6.44 (s,H-5), 3.71 (s, 7-OMe), 3.67 (s, 17-OMe), 2.72 (s, 12-NMe), 2.28 (s,16-Me), 2.20 (s, 6-Me), 0.87 (d, J=7.1 Hz, H-26);

Strain:The same as for P-22BFermentation Conditions:The same as for P-22BIsolationThe same as for P-22BBiological Activities of Safracin P-19B

Antitumor Activities Cells Lines (Mol/L) Ovary Primary Prostate SK-Breast Melanoma Endothelio NSCL Screening DU-145

OV-3 IGROV IGROV-ET SK-BR3 SK-MEL-28 HYECI A549 safracin GI50   1.70E−053.90E−06 5.42E−06 8.74E−06 7.08E−06 7.90E−06 >1.95E−05 P-19-BTGI >1.95E−05 8.06E−06 1.48E−05 >1.95E−05 1.92E−05 >1.95E−05 >1.95E−0523-OCT-02 LC50 >1.95E−051.67E−05 >1.95E−05 >1.95E−05 >1.95E−05 >1.95E−05 >1.95E−05 PrimaryLeukemia Pancreas Colon Cervix Screening K-562 PANCI HT29 LOVO LOVO-DOXHELA HELA-AFL safracin GI50 2.38E−06   1.81E−05   1.55E−05 >1.95E−05  1.44E−05 6.73E−06 4.60E−06 P-19-B TGI5.77E−06 >1.95E−05 >1.95E−05 >1.95E−05 >1.95E−05 1.61E−05 1.00E−0523-OCT-02 LC501.40E−05 >1.95E−05 >1.95E−05 >1.95E−05 >1.95E−05 >1.95E−05   1.95E−05Antimicrobial activity: On solid mediumBacillus subtilis. 10 μg/disk (6 mm diameter): NO ACTIVESpectroscopic Data:

HRFABMS m/z 495.260410 [M-H₂O+H]⁺ (calcd for C₂₇H₃₅N₄O₅ 495.260746 Δ0.3mmu); LRFABMS using m-NBA as matrix m/z (rel intensity) 495 [M-H₂O+H]⁺(13), 460 (3), 391 (2); ¹H NMR (CD₃OD, 500 MHz): 6.67 (s, H-15), 6.5 (s,H-5), 3.73 (s, 7-OMe), 3.71 (s, 17-OMe), 2.29 (s, 16-Me), 2.24 (s,6-Me), 1.13 (d, J=7.1 Hz, H-26);New Safracin Compounds Obtained by Knock Out

Strain:sac I⁻ with sacJ expression reconstitution from P. fluorescens A2-2(PM-S1-007)Fermentation Conditions:50 litres of the SAM-7 medium (50 l) composed of dextrose (3.2%),mannitol (9.6%), dry brewer's yeast (2%), ammonium sulphate (1.4%),potassium secondary phosphate (0.03%), potassium chloride (0.8%), Iron(III) chloride 6-hydrtate (0.001%), L-tyrosine (0.1%), calcium carbonate(0.8%), poly-(propylene glycol) 2000 (0.05%) and antifoam ASSAF 1000(0.2%) was poured into a jar-fermentor (Bioengineering LP-351) with 75 ltotal capacity and, after sterilization, sterile antibiotics(amplicillin 0.05 g/l and kanamycin 0.05 g/l) were added. Then, it wasinoculated with seed culture (2%) of the mutant strain PM-S1-007. Thefermentation was carried out during 89 h. under aerated and agitatedconditions (1.0 l/l/min and 500 rpm). The temperature was controlledfrom 27° C. (from the inoculation till 24 hours) to 25° C. (from 24 h tofinal process). The pH was controlled at pH 6.0 by automatic feeding ofdiluted sulphuric acid from 27 hours to final process.Isolation:

The cultured medium (45 l) thus obtained was, after removal of cells bycentrifugation, adjusted to pH 9.5 with diluted sodium hydroxide,extracted with 25 liter of ethyl acetate twice. The mixture was carriedout into an agitated-vessel at room temperature for 20 minutes. The twophases were separated by a liquid-liquid centrifuge. The organic phaseswere frozen at −20° C. and filtered for removing ice and evaporateduntil obtention of a 35 g. oil-dark-crude extract. After a 5 l. hexanetriturating, the extract (12.6 g) was purified by aflash-chromatographic column (5.5 cm diameter, 20 cm length) onsilica-normal phase, mobile phase: Ethyl acetate: MeOH: 1 L of each 1:0;20:1; 10:1; 5:1 and 7:3. 250 ml-fractions were eluted and pooleddepending of the TLC (Silica-Normal, EtOAc:MeOH 5:2, Safracin D Rf 0.2,safracin E 0.05). The fraction containing impure safracin D and E wasevaporated under high vacuum (2.2 g). An additional purification stepwas necessary to separate D and E on similar conditions (EtOAc:MeOH from1:0 to 5:1), from this, the fractions containing safracin D and E areseparate and evaporated and further purification by Sephadex LH-20column chromatography eluted with methanol.

The safracins D and E obtained were independent precipitated from CH₂Cl₂(80 ml) and Hexane (1500 ml) as a green/yellowish-dried solid (800 mgsafracin D) and (250 mg safracin E).

Biological Activities Safracin D

Antitumor Screening: Cells Lines (Mol/L) Prostate Ovary Breast MelanomaEndothelio NSCL Primary Screening DU-145

SK-OV-3 IGROV IGROV-ET SK-BR3 SK-MEL-28 HYECI A549 PM - Fernando GI505.22E−06 1.54E−06 2.69E−06 1.33E−06 4.71E−06 3.51E−06 6.04E−06 de laCalle020 19-AUG-02 TGI 9.99E−06 4.12E−06 6.02E−06 3.34E−06 7.82E−066.21E−06 1.07E−05 LC50 1.90E−05 9.78E−06 1.35E−05 9.15E−06 1.30E−051.10E−05 1.88E−05 Leukemia Pancreas Colon Cervix Primary Screening K-562PANCI HT29 LOVO LOVO-DOX HELA HELA-AFL PM - Fernando GI50 6.04E−074.77E−06 4.33E−06 6.99E−06 4.75E−06 3.76E−06 2.28E−06 de la Calle02019-AUG-02 TGI 1.16E−06 1.10E−05 1.79E−05 1.82E−05 8.85E−06 6.68E−065.24E−06 LC50 3.78E−06 >1.90E−05   >1.90E−05   >1.90E−05   1.65E−051.19E−05 1.21E−05 Secondary Evaluation (Mol/L) DNA MacromoleculesSynthesis Apoptosis Binding Cytoskeleton Secondary Screening PROTEIN DNARNA NUCLEOSOMES GEL ACTIN TUBULIN TELOMERASE PM - Fernando de la IC501.90E−05 1.52E−05 3.80E−06 2.85E−06 6.65E−06 — — Calle020 20-AUG-02Antimicrobial activity: On solid mediumBacillus subtils. 10 μg/disk (6 mm diameter): Inhibition zone: 15 mmdiameterSpectroscopic Data

ESMS: m/z 509 [M-H₂O+H]⁺; ¹H NMR (CDCl₃, 300 MHz): 6.50 (s, C-15), 4.02(s, OMe), 3.73 (s, OMe), 2.22 (s, Me), 1.85 (s, Me), 0.80 (d, J=7.2 Hz);¹³C NMR (CDCl₃, 75 MHz): 186.51, 181.15, 175.83, 156.59, 145.09, 142.59,140.78, 137.84, 131.20, 129.01, 126.88, 121.57 (2×C), 82.59, 60.92,60.69, 53.12, 21.40, 50.68, 50.22, 48.68, 40.57, 29.60, 25.01, 21.46,15.64, 8.44.

Strain:The same than safracin DFermentation Conditions:The same batch as safracin DIsolation:See safracin D conditionsBiological Activities Safracin E

Antitumor screening: Cells Lines (Mol/L) Primary Prostate Ovary BreastMelanoma Endothelio NSCL Screening DU-145

SK-OV-3 IGROV IGROV-ET SK-BR3 SK-MEL-28 HYECI A549 PM - GI50 8.34E−063.86E−06 4.50E−06 4.54E−06 5.05E−06 3.94E−06   1.96E−05 Fernando de laCalle020 TGI 1.96E−05 7.70E−06 8.85E−06 8.25E−06 9.24E−066.93E−06 >1.96E−05 19-AUG-02 LC50 >1.96E−05   1.54E−05 1.74E−05 1.49E−051.70E−05 1.22E−05 >1.96E−05 Pri mary Leukemia Pancreas Colon CervixScreening K-562 PANCI HT29 LOVO LOVO-DOX HELA HELA-AFL PM - GI504.25E−06 6.05E−06 7.89E−06 7.15E−06 5.07E−06 4.15E−06 4.03E−0

Fernando de la Calle020 TGI 8.21E−06 1.47E−05 1.96E−05 >1.96E−059.44E−06 7.29E−06 7.25E−0

19-AUG-02 LC50 1.59E−05 >1.96E−05 >1.96E−05 >1.96E−05 1.75E−05 1.28E−051.30E−0

Secondary Evaluation (Mol/L) Macromolecules Synthesis Apoptosis DNABinding Cytoskeleton Secondary Screening PROTEIN DNA RNA NUCLEOSOMES GELACTIN TUBULIN TELOMERASE PM - Fernando de la IC50 — — 1.57E−05 >1.96E−05— — — Calle020 20-AUG-02Antimicrobial activity: On solid mediumBacillus subtilis. 10 μg/disk (6 mm diameter): 9.5 mm inhibition zoneSpectroscopic Data

ESMS: m/z 511 [M+H]⁺; ¹H NMR (CDCl₃, 300 MHz): 6.51 (s, C-15), 4.04 (s,OMe), 3.75 (s, OMe), 2.23 (s, Me), 1.89 (s, Me), 0.84 (d, J=6.6 Hz); ¹³CNMR (CDCl₃, 75 MHz): 186.32, 181.28, 175.83, 156.43, 145.27, 142.75,141.05, 137.00, 132.63, 128.67, 126.64, 122.00, 120.69, 60.69, 60.21,59.12, 58.04, 57.89, 50.12, 49.20, 46.72, 39.88, 32.22, 25.33, 21.29,15.44, 8.23.

Example 5 Cross-Feeding Experiments

Heterologous Expression of Safracin Biosynthetic Precursors Genes for P2and P14 Production

In the attempt to shed light on the mechanism of the P2 and P14biosynthesis we have cloned and expressed the downstream NRPS genes todetermine their biochemical activity.

To overproduce P14, sacEFGH genes were cloned (pB7983) (FIG. 4). Tooverproduce P2 in a heterologous system, sacD to sacH genes were cloned(pB5H83)(FIG. 4). For this purpose we PCR amplified fragments harboringthe genes of interest using oligonucleotides that contain a XbaIrestriction site at the 5′ end. Oligonucleotides PFSC79(5′-CGTCTAGACACCGGCTTCATGG-3′) and PFSC83(5′-GGTCTAGATAACAGCCAACAAACATA-3′) were used to amplify sacE to sacHgenes; and oligonucleotides 5HPT1-XB (5′-CATCTAGACCGGACTGATATTCG-3′) andPFSC83 (5′-GGTCTAGATAACAGCCAACAAACATA-3′) were used to amplify sacD tosacH genes. The PCR fragments digested with XbaI were cloned into theXbaI restriction site of the pBBR1-MCS2 plasmid (Kovach et al, Gene1994, 166, 175-176). The two plasmids, pB7983 and pB5H83, were introduceseparately into three heterologous bacteria P. fluorescens (CECT 378),P. putida (ATCC12633) and P. stutzeri (ATCC 17588) by conjugation (seetable II). When culture broth of the fermentation of the transconjugantstrains was checked by HPLC analysis, big amounts of P14 compound wasvisualized in the three strains containing pB7983 plasmid, whereas bigamounts of P2 and some P14 product were observed when pB5H83 plasmid wasexpressed in the heterologa bacteria.

Cross-Feeding

As it was shown in Example 4, the sacF⁻ (PM-S1-008) and sacG⁻(PM-S1-009) mutants were not able to produce neither safracins nor P2and P14 compounds. The addition of chemically synthesized P2 to thesemutants during their fermentation yields safracin production.

Moreover, the co-cultivation of an heterologous strain of P. stutzeri(ATCC 17588) harboring plasmid pB5H83 (PM-18-004), which expressionproduces P2 and P14, with either one of the two mutants sacF⁻ and sacG⁻resulted in safracin production. The co-cultivation of an heterologousstrain P. stutzeri (ATCC 17588) harboring plasmid pB7983 (PM-18-005),which expression produces only P14, with either one of the two P.fluorescens A2-2 mutants mentioned before resulted in no safracinproduction at all. These results suggest that P14 is transformed intoP2, a molecule that can easily be transported in and out through thePseudomonas sp. cell wall and which presence it is absolutely necessaryfor the biosynthesis of safracin.

Example 6 Biological Production of New “Unnatural” Molecules

The addition of 2 g/L of an specific modified P2 derivative precursor,P3, a 3-hydroxy-5-methyl-O-methyltyrosine, to the sacF mutant(PM-S1-008) fermentation yielded two “unnatural” safracins thatincorporated the modified precursor P3 in its structure, Safracin A(OEt)and Safracin B(OEt).

Strainsaf F⁻ mutant from P. fluorescens A2-2 (PM-S1-008)Fermentation Conditions:Seed medium containing 1% glucose; 0.25% beef extract; 0.5%bacto-peptone; 0.25% NaCl; 0.8% CaCO3 was inoculated with 0.1% of afrozen vegetative stock of the microorganism, and incubated on a rotaryshaker (250 rpm) at 27° C. After 30 h of incubation, the 2% (v/v) seedculture of the mutant PM-S1-008 was transferred into 2000 ml Erlenmeyerflasks containing 250 ml of the M-16 B production medium, composed of15.2% mannitol; 3.5% Dried brewer's yeast; 1.4% (NH₄)₂ 0.001%; FeCl₃;2.6% CO₃Ca and 0.2% P3 (3-hydroxy-5-methyl-O-methyltyrosine) Thetemperature of the incubation was 27° C. from the inoculation till 40hours and then, 24° C. to final process (71 hours). The pH was notcontrolled. The agitation of the rotatory shaker was 220 rpm with 5 cmeccentricity.Isolation

4×2000/250 ml Erlenmeyer flasks were joined together (970 ml),centrifuged (12.000 rpm, 4° C., 10′, J2-21 Centrifuge BECKMAN) to removecells. The clarified broth (765 ml) was adjusted to pH 9.0 by NaOH 10%.Then, the alkali-clarified broth was extracted with 1:1 (v/v) EtOAc(×2). The organic phase was evaporated under high vacuum and agreasy-dark extract was obtained (302 mg).

This extract was washed by an hexane trituration for removing impuritiesand the solids were purified by a chromatography column using Silicanormal-phase and a mixture of Ethyl Acetate: Methanol (from 12:1 to1:1). The fractions were analyzed under UV on TLC (Silica 60, mobilephase EtOAc:MeOH 5:4. R_(f) 0.3 (Safracin B-OEt and 0.15 SafracinA-OEt). From this, safracins B OEt (25 mg) and safracin A OEt (20 mg)were obtained.

Biological Activities of Safracin B (OEt)

Antitumor Activities Cells Lines (Mol/L) Primary Prostate Ovary BreastMelanoma Endothelio Screening DU-145

SK-OV-3 IGROV IGROV-ET SK-BR3 SK-MEL-28 HYECI Safracin B Etoxi GI504.01E−07 4.84E−08 4.06E−09 6.82E−07 4.82E−09 1.69E−07 TGI1.01E−06 >1.76E−05   9.97E−09 1.19E−06 1.16E−07 4.40E−07 3-OCT-02 LC501.60E−05 8.28E−07 4.27E−06 6.37E−06 1.02E−06 1.13E−06 Primary NSCLLeukemia Pancreas Colon Cervix Screening A549 K-562 PANCI HT29 LOVOLOVO-DOX HELA HELA-AFL Safracin B Etoxi GI50 5.01E−07 3.97E−08 6.49E−072.44E−07 4.43E−07 2.09E−06 8.92E−08 7.70E−09 TGI 1.16E−06 1.08E−072.06E−06 1.39E−06 1.09E−06 9.88E−06 3.15E−07 2.74E−07 23-OCT-02 LC505.66E−06 3.69E−06 1.35E−05 >1.76E−05   >1.76E−05   >1.76E−05   1.35E−069.76E−07 Secondary Evaluation (Mol/L) DNA Secondary MacromoleculesSynthesis Apoptosis Binding Cytoskeleton Screening PROTEIN DNA RNANUCLEOSOMES GEL ACTIN TUBULIN TELOMERASE 10-OCT-02 IC50 >1.76E−051.76E−06 1.76E−07 5.28E−08 1.76E−05 — —Antimicrobial activity: On solid mediumBacillus subtilis. 10 μg/disk (6 mm diameter): 17,5 mm inhibition zoneSpectroscopic Data:

ESMS: m/z 551 [M-H₂O+H]⁺; ¹H NMR (CDCl₃, 300 MHz): 6.48 (s, H-15), 2.31(s, 16-Me), 2.22 (s, 12-NMe), 1.88 (s, 6-Me), 1.43 (t, J=6.9 Hz,Me-Etoxy), 1.35 (t, J=6.9 Hz, Me-Etoxy), 0.81 (d, J=7.2 Hz, H-26)

Strain:The same as for Safracin B (OEt)Fermentation conditions:The same as for Safracin B (OEt)Isolation:4×2000/250 ml Erlenmeyer flasks were joined together (970 ml),centrifuged (12.000 rpm, 4° C., 10′, J2-21 Centrifuge BECKMAN) to removecells. The clarified broth (765 ml) was adjusted to pH 9,0 by NaOH 10%.Then, the alkali-clarified broth was extracted with 1:1 (v/v) EtOAc(×2). The organic phase was evaporated under high vacuum and agreasy-dark extract was obtained (302 mg).This extract was washed by an hexane trituration for removing impuritiesand the solids were purified by a chromatography column using Silicanormal-phase and a mixture of Ethyl Acetate: Methanol (from 12:1 to1:1). The fractions were analysed under UV on TLC (Silica 60, mobilephase EtOAc:MeOH 5:4. Rf 0.3 Safracin B-OEt and 0.15 Safracin A-OEt).From this, safracins B OEt (25 mg) and safracin A OEt (20 mg) wereobtained.Biological Activities of Safracin A (OEt):

Antitumor Activities Cells Lines (Mol/L) Primary Prostate Ovary BreastMelanoma Endothelio Screening DU-145

SK-OV-3 IGROV IGROV-ET SK-BR3 SK-MEL-28 HYECI SafracinA Etoxi (OEt) GI502.64E−06 3.78E−07 4.92E−07 2.01E−06 5.55E−07 7.96E−07 TGI 5.39E−067.42E−07 9.28E−07 5.10E−06 1.16E−06 1.90E−06 23-OCT-02 LC50 1.10E−051.45E−06 1.76E−06 1.30E−05 5.57E−06 5.77E−06 Primary NSCL LeukemiaPancreas Colon Cervix Screening A549 K-562 PANCI HT29 LOVO LOVO-DOX HELAHELA-AFL SafracinA Etoxi (OEt) GI50 4.00E−06 3.11E−07 3.06E−06 1.97E−062.03E−06 5.72E−06 1.02E−06 7.64E−07 TGI 7.17E−06 6.66E−07 5.83E−064.41E−06 4.41E−06 9.84E−06 2.91E−06 2.32E−06 23-OCT-02 LC50 1.28E−051.51E−06 1.11E−05 9.88E−06 9.61E−06 1.69E−05 7.85E−06 6.69E−06 SecondaryEvaluation (Mol/L) Macromolecules DNA Synthesis Apoptosis BindingCytoskeleton Secondary Screening PROTEIN DNA RNA NUCLEOSOMES GEL ACTINTUBULIN TELOMERASE 10-OCT-02 IC50 — — 6.33E−06 1.81E−06 — — —Antimicrobial Activity: On Solid MediumBacillus subtilis. 10 μg/disk (6 mm diameter): 10 mm inhibition zoneSpectroscopic Data:

ESMS: m/z 553 [M+H]⁺; ¹H NMR (CDCl₃, 300 MHz): 6.48 (s, H-15), 2.33 (s,16-Me), 2.21 (s, 12-NMe), 1.88 (s, 6-Me), 1.42 (t, J=6.9 Hz, Me-Etoxy),1.34 (t, J=6.9 Hz, Me-Etoxy), 0.8 (d, J=6.9 Hz, H-26)

Example 7 Enzymatic Transformation of Safracin B into Safracin A

In order to assay the enzymatic activity of conversion of safracin Binto safracin A, a 120 hours fermentation cultures (see conditions inExample.2.Biological assay (biotest) for safracin production) ofdifferent strains were collected and centrifuged (9.000 rpm×20 min.).The strains assayed were P. fluorescens A2-2, as wild type strain, andP. fluorescens CECT378+pBHPT3 (PM-19-006), as heterologous expressionhost. Supernatant were discarded and cells were washed (NaCl 0.9%) twiceand resuspended in 60 ml phosphate buffer 100 mM pH 7.2. 20 ml from thecell suspension was distributed into three Erlenmeyer flask:

-   -   A. Cell suspension+Safracin B (400 mg/L)    -   B. Cell suspension heated at 100° C. during 10 min.+Safracin B        (400 mg/L) (negative control)    -   C. Cell suspension without Safracin B (negative control)

The biochemical reaction was incubated at 27° C. at 220 rpm and sampleswere taken every 10 min. Transformation of safracin B into safracin Awas followed by HPLC. The results clearly demonstrated that the genecloned in pBHPT3, sacH, codes for a protein responsible for thetransformation of safracin B into safracin A.

Based on this results we did an assay to find out if this same enzymewas able to recognize a different substrate such as ecteinascidin 743(ET-743) and transform this compound into Et-745 (with the C-21 hydroxymissing). The experiment above was repeated to obtain Erlenmeyer flaskscontaining:

-   -   A. Cell suspension+ET-743 (567 mg/L aprox.)    -   B. Cell suspension heated at 100° C. during 10 min.+ET-743(567        mg/L) (negative control)    -   C. Cell suspension without ET-743 (negative control)

The biochemical reaction was incubated at 27° C. at 220 rpm and sampleswere taken at o, 10 min, 1 h, 2 h, 3 h, 4 h, 20 h, 40 h, 44 h, 48 h.Transformation of ET-743 into ET-745 was followed by HPLC. The resultsclearly demonstrated that the gene cloned in pBHPT3, sacH, codes for aprotein responsible for the transformation of Et-743 into Et-745. Thisdemonstrates that this enzymes recognizes ecteinascidin as substrate andthat it can be used in the biotransformation of a broad range ofstructures.

1. A gene cluster having open reading frames which encode polypeptidessufficient to direct the synthesis of a safracin molecule.
 2. A nucleicacid sequence comprising: a) a nucleic acid sequence encoding at leastone non-ribosomal peptide synthetase which catalyse at least one step ofthe biosynthesis of safracins; b) a nucleic acid sequence which iscomplementary to the sequence in a); or c) variants or portions of thesequences of a) or b).
 3. The nucleic acid sequence according to claim 2which comprises SEQ ID NO: 1, variants or portions thereof.
 4. Thenucleic acid sequence according to claim 2 which comprises at least oneof the sacA, sacB, sacC, sacD, sacE, sacF, sacG, sacH, sacI, sacJ, orf1,orf2, orf3 or orf4 genes, including variants or portions thereof.
 5. Thenucleic acid sequence according to claim 2 wherein the nucleic acidencodes a polypeptide which is at least 30% identical in amino acidsequence to a polypeptide encoded by any of the safracin gene clusteropen reading frames sacA to sacJ and orf1 to orf4 (SEQ ID NO:1 and genesencoded in SEQ ID NO:1) or encoded by a variant or portion thereof. 6.The nucleic acid sequence according to claim 2 which encodes for any ofSacA, SacB, SacC, SacD, SacE, SacF, SacG, SacH, SacI, SacJ, Orf1, Orf2,Orf3 or Orf4 proteins (SEQ ID NO:2-15), and variants, mutants orportions thereof.
 7. The nucleic acid sequence according to claim 2which encodes a peptide synthetase, a L-Tyr derivative hidroxylase, aL-Tyr derivative methylase, a L-Tyr O-methylase, a methyl-transferase ora monooxygenase or a safracin resistance protein.
 8. The nucleic acidsequence according to any one of claims 3-6 wherein the portion is atleast 50 nucleotides in length.
 9. The nucleic acid sequence accordingto claim 8 wherein the portion is in the range between 100 to 5000nucleotides in length.
 10. The nucleic acid sequence according to claim8 wherein the portion is in the range between 100 to 2500 nucleotides inlength.
 11. A hybridization probe comprising a nucleic acid sequenceaccording to any one of the preceding claims.
 12. The hybridizationprobe according to claim 11 which comprises a sequence of at least 10nucleotide residues.
 13. The hybridization probe according to claim 11which comprises a sequence between 25 to 60 nucleotide residues.
 14. Useof a hybridization probe according to any one of claims 11-13 in thedetection of a safracin or ecteinascidin gene.
 15. The use according toclaim 14 wherein the gene detection is conducted in Ecteinascidiaturbinata.
 16. A polypeptide encoded by a nucleic acid sequence of anyone of claims 2-10.
 17. The polypeptide according to claim 16 whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:2-15.
 18. A vector comprising a nucleic acid sequence of anyone of claims 2-10.
 19. The vector according to claim 18 which is anexpression vector.
 20. The vector according to claim 18 which is acosmid.
 21. A host cell transformed with one or more of the nucleic acidsequences of any one of claims 2-10.
 22. A host cell comprising a vectorof any one of claims 18-20.
 23. The host cell according to claim 22wherein the host cell is transformed with an exogenous nucleic acidcomprising a gene cluster encoding polypeptides sufficient to direct thesynthesis of a safracin.
 24. The host cell according to claims 22 or 23which is a microorganism.
 25. The host cell according to claim 24 whichis a bacterium.
 26. A recombinant bacterial host cell in which at leasta portion of a nucleic acid sequence of any one of claims 2-10 isdisrupted to result in a recombinant host cell that produces alteredlevels of safracin compound or safracin analogue, relative to acorresponding nonrecombinant bacterial host cell.
 27. The recombinantcell of claim 26, wherein the disrupted nucleic acid sequence isendogenous.
 28. A method of producing a safracin compound or safracinanalogue comprising fermenting an organism in which the copy number ofthe gene cluster of claim 1 has been increased.
 29. A method ofproducing a safracin compound or safracin analogue comprising fermentingan organism in which expression of genes encoding polypeptidessufficient to direct the synthesis of a safracin or safracin analoguehas been modulated by manipulation or replacement of one or more genesor sequence responsible for regulating such expression.
 30. A method ofproducing a safracin compound or safracin analogue comprising contactinga compound that is a substrate for a polypeptide encoded by one or moreof the open reading frames of the safracin biosynthesis gene cluster ofclaim 1 with said polypeptide, wherein the polypeptide chemicallymodifies the compound.
 31. The method according to claims 28 or 29wherein the organism is Pseudomonas sp.
 32. A composition comprising atleast one nucleic acid sequence of any one of claims 2-10.
 33. Use of acomposition according to claim 32 for the combinatorial biosynthesis ofone or more of non-ribosomal peptide synthetases, diketopiperazine ringsand safracins.
 34. Use of P2, P14, analogs and derivatives thereof incombinatorial biosynthesis of one or more of non-ribosomal peptidesynthetases, diketopiperazine rings and safracins.
 35. A safracincompound obtainable by a method according to any of claims 28-31.
 36. Asafracin compound according to claim 35 wherein the compound has one ofthe following formulas


37. Use of a compound according to claims 35 or 36 as an antitumoragent.
 38. Use of a compound according to claims 35 or 36 in themanufacture of a medicament for the treatment of cancer.
 39. Use of acompound according to claims 35 or 36 as an antimicrobial agent.
 40. Useof a compound according to claims 35 or 36 in the manufacture of amedicament for the treatment of microbial infections.
 41. Apharmaceutical composition comprising a compound according to claims 35or 36 and a pharmaceutically acceptable diluent, carrier or excipient.42. Use of a compound according to claims 35 or 36 in the synthesis ofecteinascidin compounds.